This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Two-dimensional infrared (2D IR) spectroscopy provides a powerful method to examine protein structure and dynamics. This approach can be applied to problems that have not yet yielded to conventional methods of structural biology. For example, we recently demonstrated that 2D IR can be used to probe for tertiary contacts in a membrane protein. The protein studied in these experiments was glycophorin A (GpA), a protein that forms a constitutive homodimer in membranes. We now are extending these methods to study the transmembrane helices of integrins, proteins that are involved in signaling and cell adhesion. The intermolecular association of transmembrane domains play important roles in physiological as well as pathophysiological processes. Increasingly, it appears that the transmembrane helices of many helix-spanning proteins do far more than tether a protein in a membrane. Instead, they frequently engage in interhelical interactions that are essential for directing the assembly of protein complexes and/or signal transduction. In recent years, the GxxxG motif has emerged as an important scaffold for mediating transmembrane (TM) protein-protein interactions. The GxxxG motif (two Gly residues separated by any three residues), and "GxxxG-like" motifs (motifs in which one or both Gly residues are substituted by Ala or Ser), are often found to be important for mediating interaction of TM helices. In GpA, the GxxxG motif lies within a central in the L75IxxGVxxGVxxT87 dimer interface. The groove of the GxxxG motif, combined with the ridge of the neighboring Val residues, forms a large, almost flat surface, which serves as the central contact point of the dimer. The right-handed crossing angle about this pivot point appears to be constrained by the terminal Leu75, Ile76, and Thr87 residues, whose side chains are positioned so as to stabilize the interhelical geometry. Similar structural motifs are frequently observed in other membrane proteins. The integrin family is composed of receptors that mediate bidirectional communication between cells and between the cell and the extracellular matrix. The integrins are type I integral membrane proteins with a conserved GxxxG-like motif in their TM domains, which are likely to be functionally involved in the oligomerization events that are critical for signaling. Mutagenesis studies of integrin TM domains have shown that GxxxG and GxxxG-like motifs figure largely in the structures of the various interfaces, but there have been no high-resolution structural studies of this motif, despite very extensive efforts in the area of NMR and crystallography. Thus, IR will provide an important tool to examine both the dynamics as well the structures of integrin TM helix-helix interactions.